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Fe3O4@Au nanoparticles as a means of signal enhancement in surface plasmon resonance spectroscopy for thrombin detection
Sensors and Actuators B 212 (2015) 505–511
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Fe3 O4 @Au nanoparticles as a means of signal enhancement in surface plasmon resonance spectroscopy for thrombin detection Hongxia Chen a,∗ , Fangjie Qi a , Hongjian Zhou b , Shengsong Jia a , Yanmin Gao a , Kwangnak Koh c , Yongmei Yin d,∗∗ a
Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, PR China Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735, Republic of Korea c Institute of General Education, Pusan National University, Busan 609-735, Republic of Korea d Department of Oncology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, PR China b
a r t i c l e
i n f o
Article history: Received 18 November 2014 Received in revised form 13 January 2015 Accepted 5 February 2015 Available online 19 February 2015 Keywords: Thrombin Aptamer Surface plasmon resonance Signal amplication Gold capped magnetic nanoparticles
a b s t r a c t The core-shell gold capped magnetic nanoparticles (GMPs) have been receiving increasing attention because of its optical and magnetic properties. In order to evaluate the ability of GMPs as a means of signal enhancement in surface plasmon resonance (SPR) spectroscopy, a sandwich SPR sensor is constructed by using thrombin as a model analyte. In this design, thrombin is captured by thrombin aptamer 1 (Apt1) and sensitively detected through addition of GMPs-Apt2 conjugates. Compared with gold nanoparticles (GNPs) and magnetic nanoparticles (MNPs), GMPs-Apt2 conjugates result in a significant SPR angle increase, which is mainly attributed by the larger mass and higher refractive index of the GMP nanoparticles. Therefore, the detection limit can be achieved as low as 0.1 nM. Since GMP may have high stability and biocompatibility, it can be a useful sandwich element for sensor fabrication and an excellent amplification reagent for SPR measurement. This work may also afford a new model to improve the sensitivity and selectivity of SPR biosensors in protein detection and disease diagnosis. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Nanomaterials have attracted more and more attention in the field of biosensor on the basis of their unique optical, electronic, and catalytic properties [1–5]. After introduction of nanomaterials, the performance of biosensors can be improved obviously, which may also promote the research and development of novel kind of biosensors. Surface plasmon resonance (SPR) biosensor is a rising technology in the field of biomarker detection. The SPR system allows a label-free, real-time, highly sensitive monitoring of the whole assay process [6,7]. In the meantime, it has also received great interest to select a kind of nanomaterial which can lead to SPR signal amplification, and finally improve the sensitivity of SPR biosensor. The application of gold nanoparticles (GNPs) [8–10] and magnetic nanoparticles (MNPs) [11–13] in the SPR optical biosensor has been attracted increasing attention. Lyon et al. have applied GNPs as the signal-amplifying label in the sandwich structure immunoassay
∗ Corresponding author. Tel.: +86 21 6613 7539. ∗∗ Corresponding author. Tel.: +86 25 68136043. E-mail addresses: [email protected] (H. Chen), [email protected] (Y. Yin). http://dx.doi.org/10.1016/j.snb.2015.02.062 0925-4005/© 2015 Elsevier B.V. All rights reserved.
[14]. Ascribed to their biocompatibility and high refractive index, the SPR signal after being labeled with GNPs increases twenty times than that of non-labelling. Meanwhile, Wang et al. have revealed that electromagnetic field (EMF) may enhance the signal of sandwich immunosensor labeled with MNPs to four orders of magnitudes [13]. The grating SPR sensor fabricated by Wang and his co-workers has also been applied in the detection of HCG. However, there has no report about using MNPs covered with gold colloid (GMPs) to fabricate the sensitive SPR sensor. In this work, GMPs are utilized as a means of signal enhancement in surface plasmon resonance spectroscopy, and thrombin is employed as a model protein here. Thrombin has two aptamers (Apt1, 15 bases sequence, and Apt2, 29 bases sequence) [15–18], which are used to recognize the different parts of thrombin to form a sandwich structure. As shown in Fig. 1, thiolated 15-mer Apt1 with polythymine (T10) is designed in this study and immobilized on the gold disk through covalent binding between the thiol group of Apt1 and Au. The T10 part of Apt1 acts as a spacer to provide enough combination space. Mercaptohexanol (MCH) is used in the next step to backfill the gold disk so as to resist nonspecific binding and obtain well aligned aptamer monolayers [19]. In the presence of thrombin, Apt1 can recognize it and form G-quarter structure. After that, AuNPs-Apt2 conjugates, MNPs-Apt2 conjugates and
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Fourier transform infrared (FT-IR) spectroscopy (JASCO, FTIR6300, Japan). The immobilization of the thiolated 29-mer Apt2 onto GNPs was carried out by the following steps. Briefly, 3 mL of GNPs was mixed with 10 L of 100 M Apt for 24 h. The resulting solution was then centrifuged at 12000 rpm for 20 min and the sediment was washed with Tris-HCl buffer and resuspended in 500 L of Tris–HCl buffer [23]. Then, the GNPs-Apt2 solution was stored at 4 ◦ C. 2.3. The MNPs synthesis and immobilization of Apt2 onto MNPs
Fig. 1. Scheme for the formation of sandwich structure on the SPR chip surface.
GMPs-Apt2 conjugates are added into the SPR chamber separately to form an Apt1/thrombin/NPs-Apt2 sandwich structure. Experimental results revealed that this sandwich SPR sensor fabricated with GMPs may maked use of the two kinds of nanoparticles, and a better sensitivity can be achieved. 2. Experimental 2.1. Materials Hydrogen tetrachloroaurate (HAuCl4 ), trisodium citrate, 6mercaptohexanol (MCH), thrombin, N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbo-diimide hydrochloride (EDC), tris(2-carboxyethyl)phosphine (TCEP) were purchased from Sigma-Aldrich (St. Louis, MO). Albumin from bovine serum (BSA) was obtained from Zhaorui biotech Co. Ltd. (Shanghai, China). The oligonucleotide with the following sequence: thiolated 15-mer Apt1 with T10 tail, 5 SH-(CH2 )6 -TTTTTTTTTTGGTTGGTGTGGTTGG; thiolated or amino-modified 29-mer Apt2 with polyT 20 tail, 5 SH-(CH2 )6 -TTTTTTTTTTTTTTTTTTTTAGT-CCGTGGTAGGGCAGGTTGGGGTGACT and 5 NH2 -(CH2 )6 -TTTTTTTTTTTTT-TTTTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT used in this study were purchased from Shanghai Sangon Biological Engineering Technology & Service Co., Ltd. Buffer for washing and binding: Tris-Hcl buffer (pH 7.4, 50 mM Tris, 100 mM NaCl, 5 mM KCl, 1 mM MgCl2 , and 5 mM CaCl2 ); PBS buffer (pH 7.4, 150 mM NaCl, 20 mM Na2 HPO4 , 3 mM NaH2 PO4 ). Milli-Q grade (18.2 m cm−1 ) water was used for preparation of samples and buffer solutions. 2.2. The GNPs synthesis and immobilization of Apt2 onto GNPs GNPs used in this study were prepared by citrate reduction of HAuCl4 in aqueous solution [20–22]. Firstly, 95.5 mL of ultrapure water was heated to boiling and vigorously stirred in a roundbottom flask. Following this, 1 mL of 1% HAuCl4 was added to the vortex of the boiling solution about 1 min. Then 3.5 mL of 1% trisodium citrate was quickly added to the solution. The color of the solution changed from grey to wine red, indicating the formation of gold nanoparticles. After boiling for an additional 15 min, the solution was cooled to room temperature with a continuous stirring for another 45 min. The colloids were stored in dark glass bottles at 4 ◦ C for further use. The prepared GNPs, MNPs and GMPs were characterized using UV–Vis spectroscopy. Morphologies and sizes of the nanoparticles were characterized by HR-TEM (JEOL, JEM-3010, Japan) and the surface modification was monitored by
Under nitrogen atmospheric, 1.622 g of FeCl3 ·6H2 O and 0.9941 g of FeCl2 ·4H2 O was dissolved into 40 mL of ultrapure water with constant mechanical stirring. After the reagents had completely dissolved, 5 mL of ammonia solution (28%, w/v %) was quickly added to the reaction mixture. Ten minutes later, we added 4.4 g of trisodium citrate and the reaction temperature was raised to 90 ◦ C, and maintained at that temperature with continuous stirring for 30 min. A black precipitate was obtained by cooling the reaction mixture to room temperature [24]. The precipitate was then thoroughly rinsed with ethanol for three times. During rinsing, samples were separated from the supernatant using a permanent magnet. After discarding the supernatant, samples were dried under vacuum and stored at 4 ◦ C for further use. To prepare MNPs-Apt2 conjugates, 3 mg of MNPs were washed with TE buffer (pH 7.5, 10 mM Tris, 1 mM EDTA) for three times and resuspended in 300 L of TE buffer, then 300 L of NHS (0.1 M) and 300 L of EDC (0.4 M) were mixed with the solution for 30 min at 37 ◦ C to activate the carboxyl group on the surface of MNPs. After that, MNPs were washed with TE buffer for three times and resuspended in 300 L of TE buffer. Then 300 L of 1 M amino-modified 29-mer Apt2 was added for 2 h at 37 ◦ C. At last, MNPs-Apt2 conjugates were washed with TE buffer for three times and resuspended in 300 L of TE buffer. 2.4. The GMPs synthesis and immobilization of Apt2 onto GMPs HAuCl4 (20 mL, 0.5 mM) was heated to boiling and vigorously stirred in a round-bottom flask. Then rapid addition 10 mL of the MNPs solution prepared from the previous step resulted in a continuous color change from brown to burgundy. The ratios of reactive volumes between Fe3 O4 and HAuCl4 solution were 1:2. Stirring continued for 10 min after the color change ceased [25]. The solution was cooled to room temperature with a continuous stirring for another 45 min. The Au coated MNPs were centrifuged three times at 6500 rpm for 30 min. After that, the product was washed with ultrapure water and re-dispersed in ultrapure water. For the preparation of the GMPs-Apt2 conjugates, 50 L GMPs were washed three times with Tris-HCl buffer and resuspended in 495 L of Tris-HCl buffer, then ultrasonicated for 30 min. 5 L of thiolated 29-mer Apt2 (100 M) was added into the above solution, and the mixture was stirred for 2 h at room temperature. After magnetic separation, unbounded aptamer was removed, then the GMPs-Apt2 conjugates was washed three times with 500 L of TrisHCl buffer, and then 500 L of 1 mM MCH was added and incubated for 40 min. After washing and magnetic separation, the conjugates were resuspended in 500 L of Tris-HCl buffer and stored at 4 ◦ C. 2.5. The fabrication of thrombin aptasensor and SPR detection The Apt1 was designed to contain a thiol group in 5 terminal. The thiol group of aptamer is used for covalent coupling to the Au surface. To immobilize Apt1 on the gold surface, a gold chip was purchased from Metrohm AG (Hersau Switzerland) as a substrate for immobilization of aptamer. Before the immobilization, the gold chip was first soaked in piranha solution (98% H2 SO4 :30%
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Fig. 2. TEM images of GNPs (A), MNPs (B), GMPs (C) and FTIR spectra of citrate and GMPs (D).
H2 O2 = 3:1, v/v) for 5 min to eliminate the adsorbed material and then rinsed with double distilled water [25]. Then, the prepared gold chip was fixed on the SPR instrument. The immobilization of Apt1 on the gold chip was performed at 27 ◦ C for 16 h by incubation with a 1 M Apt1 solution (dissolved in TE buffer, pH 7.5). After the immobilization, the gold chip was rinsed with distilled water (DW) and backfilled with 1 mM MCH for 1 h in the absence of light. Then gold chip was flushed by ultrapure water until a stable SPR baseline was acquired. 100 nM of thrombin was added to react with Apt1 to form specific binding. After that, gold chip was rinsed with ultrapure water to remove unbounded thrombin. Finally, the diluted nanoparticles and aptamer conjugates were added to form Apt1/thrombin/NPs-Apt2 conjugates. The whole process was monitored by SPR spectroscopy in real-time. SPR measurements were performed by an Autolab ESPRIT system (Eco Chemie B.V., the Netherlands) equipped with a 670 nm monochromatic p-polarized light source.
3. Results and discussion 3.1. Characterization of synthesized nanoparticles GNPs, MNPs and GMPs have been characterized by transmission electron microscopy (TEM) and the results are shown in Fig. 2. The TEM micrograph shows the shape of GNPs (Fig. 2A), and the size of GNPs is about 16 nm in diameter. Fig. 2B provides the TEM image of the prepared MNPs, which clearly shows that the prepared MNPs are almost spherical in shape and have a size of about 15 nm. In addition, Fig. 2C shows the TEM micrograph of GMPs, and the
size of GMPs is about 25 nm in diameter. The GMPs and sodium citrate were also measured through the FT-IR spectra, as shown in Fig. 2D. The 1530 cm−1 peak attributed to the C O stretching vibration from the COOH group of citrate shifts to an intense band at about 1525 cm−1 for GMPs. It demonstrated citrate’s bonding on the surface of GMPs by chemisorptions. 3.2. Characterizations of the nanoparticle-Apt2 conjugates Nanoparticles, aptamer and NPs-Apts conjugates were characterized by UV–vis spectroscopy and the results are shown in Fig. 3. The formation of conjugates was characterized by the UV–vis absorption spectrum. Fig. 3a, c and e recorded the UV–vis absorption of GNPs, MNPs and GMPs respectively. After coated with Apt2, UV–vis curves showed red shifts for all three particles. The bare GNPs exhibited a characteristic absorption peak at 518 nm (Fig. 3a), which related to the surface plasmon resonance of nanosized Au. After coated with Apt2, UV–vis absorption was observed at 520 nm due to the formation of GNPs-Apt2 (curve b). Curve c shows the UV–vis absorption spectrum of MNPs with an absorption peak at 223 nm. After the incubation of Apt2, the absorption peak shifts to 247 nm, indicating that Apt2 has been successful associated to MNPs. Curves e and f show the absorption peak of GMPs at 548 nm and new absorption peak of GMPs-Apt2 at 559 nm, it also demonstrate the Apt2 was successful bound onto GMPs. The curve g is the UV–vis absorption spectrum of Apt2 and the absorption peak of Apt2 was appeared at 260 nm, which correspond to the UV–vis absorption peak of DNA. All these results verify that Apt2 has been immobilized on the nanoparticles successfully.
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Fig. 3. UV–vis spectra of GNPs (a, black), GNPs-Apt2 conjugates (b, red), MNPs (c, blue), MNPs-Apt2 conjugates (d, green), GMPs (e, pink), GMPs-Apt2 conjugates (f, dark green) and Apt2 (g, dark blue). (For interpretation of the color information in this figure legend, the reader is referred to the web version of the article.)
3.3. Immobilization of Apt1 on gold chip surface Apt1 was immobilized on the gold surface by self-assembly via the covalent bond between Au and the thiol group of aptamer as described elsewhere [26]. SPR was employed to characterize the
Fig. 4. Sensorgram of SPR angle shifts according to the immobilization of Apt1 and MCH on chip surface.
immobilization. The interaction between Au and Apt1 was monitored by observing changes in the resonant angle. The SPR angle increase in millidegrees is used as a response unit to quantify the binding of macromolecules to the sensor surface. A change of 120 millidegrees represents a surface change of approximately 1 ng mm−2 as described in previous reports [27–29]. To obtain well aligned aptamer monolayers [30], 1 mM MCH was injected into two
Fig. 5. The sensogram of SPR signal amplification processes with three kinds of nanoparticles GNPs (A), MNPs (B), GMPs (C) and histogram of the comparison of three nanoparticles (D). The black line is experimental group. The red line is control group, which is treated with BSA instead of thrombin.
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Fig. 6. AFM images of the bare gold film (A) and sequentially treated with Apt1/thrombin/GNPs-Apt2 (B), Apt1/thrombin/MNPs-Apt2 (C), and Apt1/thrombin/GMPs-Apt2 (D).
channels for 1 h. As shown in Fig. 4, an increase in the SPR angle was observed, the adsorption of aptamer on SPR gold chip results in a big angle shift of 789 millidegrees. The surface coverage of aptamer on the gold chip is 6.575 ng mm−2 as calculated. 3.4. SPR signal enhancement by three NPs-Apt2 Aptamer prefers to adopt G-quarter structure when binding with thrombin [31]. Incubation conditions, such as pH value, temperature and so on, have an impact on the formation of the G-quarter structure [32]. Thrombin was dissolved in Tris buffer (pH 6.2, 100 mM MES, 50 mM Tris, 40 mM KCl, 0.05% Triton X-100, and 1% DMSO) and injected into the SPR chamber for 30 min at room temperature (Fig. 5, experimental group, black line). In order to confirm the specific binding of Apt1 and thrombin, a two channel SPR system was used. The control group (Fig. 5, red line) was injected with bovine serum albumin (BSA) solution with the corresponding concentrations. As shown in Fig. 5, the SPR angle was increased evidently with the injection of thrombin. However, when BSA with same concentration was injected into the SPR chamber, no obvious SPR signal was observed. This result confirms that the Apt1 has a sufficient specificity and thrombin can be identified
with high selectivity. After the incubation of thrombin, the conjugate of nanoparticles and Apt2 was injected into SPR chamber for 15 min. The increase of SPR angle is obvious difference with the injection of different NPs-Apt2 conjugates. Fig. 5 shows SPR angel shifts after the injection of GNPs-Apt2, MNPs-Apt2 and GMPsApt2 conjugates are 313, 431and 604 millidegrees, respectively. The amplification GMPs-Apt2 conjugate for SPR signal is 1.93 and 1.40 times of that of GNPs-Apt2 and MNPs-Apt2 respectively. However, even after the injection of NPs-Apt2 into the control group, the SPR signal is kept stable. These results demonstrate the formation of Apt1/thrombin/NPs-Apt2 sandwich structure successfully. As shown in Fig. 5D, compared with GNPs and MNPs, the signal amplification of GMPs for SPR spectroscopy is more sensitive. This enhancement was mainly attributed by the larger mass and higher refractive index of the GMP nanoparticles. Therefore, we choose GMPs-Apt2 conjugates as signal amplification element in following thrombin detection. To further confirm the binding of NPs-Apt2 onto the thrombin modified chip surface, we carried out AFM image characterization. Fig. 6A shows the surface geometry of the bare gold film. After the immobilization of Apt1 and the formation of sandwich structure, the images reveal that the height increased from 5 to 35 nm
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Fig. 7. Direct detection of thrombin (A) and its comparison with amplification strategy using GMPs at variable concentrations of thrombin (B).
(Fig. 6B–D). Among three NPs, GNP and MNP show high aggregation which may due to the lower stability after modified with Apt2. The monodispersed GMP shows good uniform and lower aggregation on the sensor chip surface.
great potential of GMPs in the field of protein detection and disease diagnosis.
Acknowledgements 3.5. Thrombin detection via Apt-immobilized GMPs The SPR detection of thrombin was preformed to evaluate the sensing performance of the Apt-immobilized GMPs as amplification reagent. It could be clearly seen from the curves in Fig. 7A that the SPR angel shifts gradually increase with the concentration of thrombin concentration increasing from 10 to 100 nM. However, when the concentration of thrombin concentration is lower than 10 nM, it is very difficult to discriminate the concentration of thrombin by SPR spectroscopy due to its low molecular weight. Obviously, this detection limit is not enough for the detection of thrombin due to the trace amount of thrombin existed in serum. In order to detect thrombin at low concentrations, GMPs-Apt2 conjugates were used as an amplification reagent. As shown in Fig. 7B, SPR angel shifts resulted in the binding of GMPs-Apt2 conjugates greatly increase with the thrombin concentrations in the range of 0.1–100 nM (black curve). Thrombin detection using GMPs-Apt2 conjugates for signal enhancement shows good linearity from 0.1 to 100 nM with R2 = 0.98. The limit of detection (LOD) was calculated to be 0.60 nM (LOD = 3.3 × standard deviation/slope). At the concentration of 100 nM, the SPR angle shift is enlarged for 5 times comparing with that of control group without GMPs-Apt2. Therefore, the increase in the SPR signal obtained for the experimental group can definitely own to GMPs-Apt2 conjugates. The whole trends of the experimental group and the control group verified that GMPs is an outstanding means of signal amplification for SPR. Aptamer modified GMPs can result in significant enhancement of the SPR sensor’s sensitivity.
4. Conclusions In summary, the monodispersed core/shell Fe3 O4 /Au magnetic nanoparticles (GMPs) have been synthesized. To evaluate the practicability of using GMPs in enhancing SPR signal for biosensing, thrombin is used as the model analyte to construct SPR-based sandwich biosensor. The experimental results demonstrate that the addition of GMPs-Apt2 conjugates results in a big SPR angle change, indicating that GMPs possess of the ability of enhancing the sensitivity of SPR sensor as an amplification reagent. It shows
This work is supported by the National Natural Science Foundation of China (Grant Nos. 61275085, 31100560).
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Biographies Hongxia Chen received her B.S. degree in chemistry in 1996 from Zhengzhou University, China, and MS degree in 2003 and Ph.D. in 2008 from Pusan National University, Korea. Dr. Chen is an associate professor in School of Life Sciences, Shanghai University. Her current research interests include optical sensors, biochip, bioanalytical nanochemistry and nanotoxicology. Fangjie Qi received his B.S. degree from school of Kewen in 2012 from Jiangsu Normal University, China. He is a master course student in School of Life Sciences, Shanghai University. His current research interests are biosensor and cancer cell detection. Hongjian Zhou received his B.S. degree in chemistry in 2007 from Nanyang Institute of Technology, China, and MS degree in Materials physics and chemistry in 2010 from Jiangsu University of Science and Technology, China. He got his Ph.D. degree in Nano Fusion Technology in 2014 from Pusan National University. Now he is a Korean National Research Foundation (KNRF)-funded research fellow in Department of Cogno-Mechatronics Engineering, Pusan National University. His current research interest includes synthesis of multifunctional nanoparticles and biosensor applications. He is also interested in surface modification of nanocomposites for biocompatibility and biodegradability in bio-nano-medical applications. Shengsong Jia received her B.S. degree in 2013 from School of Xinke, Hennan Institude of Science and Technology, China. She is a master course student in School of Life Sciences, Shanghai University. Her current research interests are biosensor and small molecule detection. Yanmin Gao received her B.S. degree in 2013 from school of biological science, Nanyang Normal University, China. She is a master course student in School of Life Sciences, Shanghai University. Her current research interests are biosensor and cancer cell detection. Kwangnak Koh received M.S. degree in 1992 from Pusan National University and Ph.D. in 1995 in supramolecular engineering from Kyushu University, Japan. He is a professor in institute of general education, Pusan National University, Korea. His research interests include biochip, supramolecular engineering, bioanalytical nanochemistry and bionanomaterials. Yongmei Yin received her bachelor, master and Ph.D. degree at Nanjing Medical University in 1991, 2001 and 2008, respectively. She is currently the vice-Dean of the First Affiliated Hospital of Nanjing Medical University, and the vice-director of the Department of Oncology at this Hospital.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Fe3 O4 @Au nanoparticles as a means of signal enhancement in surface plasmon resonance spectroscopy for thrombin detection Hongxia Chen a,∗ , Fangjie Qi a , Hongjian Zhou b , Shengsong Jia a , Yanmin Gao a , Kwangnak Koh c , Yongmei Yin d,∗∗ a
Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, PR China Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735, Republic of Korea c Institute of General Education, Pusan National University, Busan 609-735, Republic of Korea d Department of Oncology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, PR China b
a r t i c l e
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Article history: Received 18 November 2014 Received in revised form 13 January 2015 Accepted 5 February 2015 Available online 19 February 2015 Keywords: Thrombin Aptamer Surface plasmon resonance Signal amplication Gold capped magnetic nanoparticles
a b s t r a c t The core-shell gold capped magnetic nanoparticles (GMPs) have been receiving increasing attention because of its optical and magnetic properties. In order to evaluate the ability of GMPs as a means of signal enhancement in surface plasmon resonance (SPR) spectroscopy, a sandwich SPR sensor is constructed by using thrombin as a model analyte. In this design, thrombin is captured by thrombin aptamer 1 (Apt1) and sensitively detected through addition of GMPs-Apt2 conjugates. Compared with gold nanoparticles (GNPs) and magnetic nanoparticles (MNPs), GMPs-Apt2 conjugates result in a significant SPR angle increase, which is mainly attributed by the larger mass and higher refractive index of the GMP nanoparticles. Therefore, the detection limit can be achieved as low as 0.1 nM. Since GMP may have high stability and biocompatibility, it can be a useful sandwich element for sensor fabrication and an excellent amplification reagent for SPR measurement. This work may also afford a new model to improve the sensitivity and selectivity of SPR biosensors in protein detection and disease diagnosis. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Nanomaterials have attracted more and more attention in the field of biosensor on the basis of their unique optical, electronic, and catalytic properties [1–5]. After introduction of nanomaterials, the performance of biosensors can be improved obviously, which may also promote the research and development of novel kind of biosensors. Surface plasmon resonance (SPR) biosensor is a rising technology in the field of biomarker detection. The SPR system allows a label-free, real-time, highly sensitive monitoring of the whole assay process [6,7]. In the meantime, it has also received great interest to select a kind of nanomaterial which can lead to SPR signal amplification, and finally improve the sensitivity of SPR biosensor. The application of gold nanoparticles (GNPs) [8–10] and magnetic nanoparticles (MNPs) [11–13] in the SPR optical biosensor has been attracted increasing attention. Lyon et al. have applied GNPs as the signal-amplifying label in the sandwich structure immunoassay
∗ Corresponding author. Tel.: +86 21 6613 7539. ∗∗ Corresponding author. Tel.: +86 25 68136043. E-mail addresses: [email protected] (H. Chen), [email protected] (Y. Yin). http://dx.doi.org/10.1016/j.snb.2015.02.062 0925-4005/© 2015 Elsevier B.V. All rights reserved.
[14]. Ascribed to their biocompatibility and high refractive index, the SPR signal after being labeled with GNPs increases twenty times than that of non-labelling. Meanwhile, Wang et al. have revealed that electromagnetic field (EMF) may enhance the signal of sandwich immunosensor labeled with MNPs to four orders of magnitudes [13]. The grating SPR sensor fabricated by Wang and his co-workers has also been applied in the detection of HCG. However, there has no report about using MNPs covered with gold colloid (GMPs) to fabricate the sensitive SPR sensor. In this work, GMPs are utilized as a means of signal enhancement in surface plasmon resonance spectroscopy, and thrombin is employed as a model protein here. Thrombin has two aptamers (Apt1, 15 bases sequence, and Apt2, 29 bases sequence) [15–18], which are used to recognize the different parts of thrombin to form a sandwich structure. As shown in Fig. 1, thiolated 15-mer Apt1 with polythymine (T10) is designed in this study and immobilized on the gold disk through covalent binding between the thiol group of Apt1 and Au. The T10 part of Apt1 acts as a spacer to provide enough combination space. Mercaptohexanol (MCH) is used in the next step to backfill the gold disk so as to resist nonspecific binding and obtain well aligned aptamer monolayers [19]. In the presence of thrombin, Apt1 can recognize it and form G-quarter structure. After that, AuNPs-Apt2 conjugates, MNPs-Apt2 conjugates and
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Fourier transform infrared (FT-IR) spectroscopy (JASCO, FTIR6300, Japan). The immobilization of the thiolated 29-mer Apt2 onto GNPs was carried out by the following steps. Briefly, 3 mL of GNPs was mixed with 10 L of 100 M Apt for 24 h. The resulting solution was then centrifuged at 12000 rpm for 20 min and the sediment was washed with Tris-HCl buffer and resuspended in 500 L of Tris–HCl buffer [23]. Then, the GNPs-Apt2 solution was stored at 4 ◦ C. 2.3. The MNPs synthesis and immobilization of Apt2 onto MNPs
Fig. 1. Scheme for the formation of sandwich structure on the SPR chip surface.
GMPs-Apt2 conjugates are added into the SPR chamber separately to form an Apt1/thrombin/NPs-Apt2 sandwich structure. Experimental results revealed that this sandwich SPR sensor fabricated with GMPs may maked use of the two kinds of nanoparticles, and a better sensitivity can be achieved. 2. Experimental 2.1. Materials Hydrogen tetrachloroaurate (HAuCl4 ), trisodium citrate, 6mercaptohexanol (MCH), thrombin, N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbo-diimide hydrochloride (EDC), tris(2-carboxyethyl)phosphine (TCEP) were purchased from Sigma-Aldrich (St. Louis, MO). Albumin from bovine serum (BSA) was obtained from Zhaorui biotech Co. Ltd. (Shanghai, China). The oligonucleotide with the following sequence: thiolated 15-mer Apt1 with T10 tail, 5 SH-(CH2 )6 -TTTTTTTTTTGGTTGGTGTGGTTGG; thiolated or amino-modified 29-mer Apt2 with polyT 20 tail, 5 SH-(CH2 )6 -TTTTTTTTTTTTTTTTTTTTAGT-CCGTGGTAGGGCAGGTTGGGGTGACT and 5 NH2 -(CH2 )6 -TTTTTTTTTTTTT-TTTTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT used in this study were purchased from Shanghai Sangon Biological Engineering Technology & Service Co., Ltd. Buffer for washing and binding: Tris-Hcl buffer (pH 7.4, 50 mM Tris, 100 mM NaCl, 5 mM KCl, 1 mM MgCl2 , and 5 mM CaCl2 ); PBS buffer (pH 7.4, 150 mM NaCl, 20 mM Na2 HPO4 , 3 mM NaH2 PO4 ). Milli-Q grade (18.2 m cm−1 ) water was used for preparation of samples and buffer solutions. 2.2. The GNPs synthesis and immobilization of Apt2 onto GNPs GNPs used in this study were prepared by citrate reduction of HAuCl4 in aqueous solution [20–22]. Firstly, 95.5 mL of ultrapure water was heated to boiling and vigorously stirred in a roundbottom flask. Following this, 1 mL of 1% HAuCl4 was added to the vortex of the boiling solution about 1 min. Then 3.5 mL of 1% trisodium citrate was quickly added to the solution. The color of the solution changed from grey to wine red, indicating the formation of gold nanoparticles. After boiling for an additional 15 min, the solution was cooled to room temperature with a continuous stirring for another 45 min. The colloids were stored in dark glass bottles at 4 ◦ C for further use. The prepared GNPs, MNPs and GMPs were characterized using UV–Vis spectroscopy. Morphologies and sizes of the nanoparticles were characterized by HR-TEM (JEOL, JEM-3010, Japan) and the surface modification was monitored by
Under nitrogen atmospheric, 1.622 g of FeCl3 ·6H2 O and 0.9941 g of FeCl2 ·4H2 O was dissolved into 40 mL of ultrapure water with constant mechanical stirring. After the reagents had completely dissolved, 5 mL of ammonia solution (28%, w/v %) was quickly added to the reaction mixture. Ten minutes later, we added 4.4 g of trisodium citrate and the reaction temperature was raised to 90 ◦ C, and maintained at that temperature with continuous stirring for 30 min. A black precipitate was obtained by cooling the reaction mixture to room temperature [24]. The precipitate was then thoroughly rinsed with ethanol for three times. During rinsing, samples were separated from the supernatant using a permanent magnet. After discarding the supernatant, samples were dried under vacuum and stored at 4 ◦ C for further use. To prepare MNPs-Apt2 conjugates, 3 mg of MNPs were washed with TE buffer (pH 7.5, 10 mM Tris, 1 mM EDTA) for three times and resuspended in 300 L of TE buffer, then 300 L of NHS (0.1 M) and 300 L of EDC (0.4 M) were mixed with the solution for 30 min at 37 ◦ C to activate the carboxyl group on the surface of MNPs. After that, MNPs were washed with TE buffer for three times and resuspended in 300 L of TE buffer. Then 300 L of 1 M amino-modified 29-mer Apt2 was added for 2 h at 37 ◦ C. At last, MNPs-Apt2 conjugates were washed with TE buffer for three times and resuspended in 300 L of TE buffer. 2.4. The GMPs synthesis and immobilization of Apt2 onto GMPs HAuCl4 (20 mL, 0.5 mM) was heated to boiling and vigorously stirred in a round-bottom flask. Then rapid addition 10 mL of the MNPs solution prepared from the previous step resulted in a continuous color change from brown to burgundy. The ratios of reactive volumes between Fe3 O4 and HAuCl4 solution were 1:2. Stirring continued for 10 min after the color change ceased [25]. The solution was cooled to room temperature with a continuous stirring for another 45 min. The Au coated MNPs were centrifuged three times at 6500 rpm for 30 min. After that, the product was washed with ultrapure water and re-dispersed in ultrapure water. For the preparation of the GMPs-Apt2 conjugates, 50 L GMPs were washed three times with Tris-HCl buffer and resuspended in 495 L of Tris-HCl buffer, then ultrasonicated for 30 min. 5 L of thiolated 29-mer Apt2 (100 M) was added into the above solution, and the mixture was stirred for 2 h at room temperature. After magnetic separation, unbounded aptamer was removed, then the GMPs-Apt2 conjugates was washed three times with 500 L of TrisHCl buffer, and then 500 L of 1 mM MCH was added and incubated for 40 min. After washing and magnetic separation, the conjugates were resuspended in 500 L of Tris-HCl buffer and stored at 4 ◦ C. 2.5. The fabrication of thrombin aptasensor and SPR detection The Apt1 was designed to contain a thiol group in 5 terminal. The thiol group of aptamer is used for covalent coupling to the Au surface. To immobilize Apt1 on the gold surface, a gold chip was purchased from Metrohm AG (Hersau Switzerland) as a substrate for immobilization of aptamer. Before the immobilization, the gold chip was first soaked in piranha solution (98% H2 SO4 :30%
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Fig. 2. TEM images of GNPs (A), MNPs (B), GMPs (C) and FTIR spectra of citrate and GMPs (D).
H2 O2 = 3:1, v/v) for 5 min to eliminate the adsorbed material and then rinsed with double distilled water [25]. Then, the prepared gold chip was fixed on the SPR instrument. The immobilization of Apt1 on the gold chip was performed at 27 ◦ C for 16 h by incubation with a 1 M Apt1 solution (dissolved in TE buffer, pH 7.5). After the immobilization, the gold chip was rinsed with distilled water (DW) and backfilled with 1 mM MCH for 1 h in the absence of light. Then gold chip was flushed by ultrapure water until a stable SPR baseline was acquired. 100 nM of thrombin was added to react with Apt1 to form specific binding. After that, gold chip was rinsed with ultrapure water to remove unbounded thrombin. Finally, the diluted nanoparticles and aptamer conjugates were added to form Apt1/thrombin/NPs-Apt2 conjugates. The whole process was monitored by SPR spectroscopy in real-time. SPR measurements were performed by an Autolab ESPRIT system (Eco Chemie B.V., the Netherlands) equipped with a 670 nm monochromatic p-polarized light source.
3. Results and discussion 3.1. Characterization of synthesized nanoparticles GNPs, MNPs and GMPs have been characterized by transmission electron microscopy (TEM) and the results are shown in Fig. 2. The TEM micrograph shows the shape of GNPs (Fig. 2A), and the size of GNPs is about 16 nm in diameter. Fig. 2B provides the TEM image of the prepared MNPs, which clearly shows that the prepared MNPs are almost spherical in shape and have a size of about 15 nm. In addition, Fig. 2C shows the TEM micrograph of GMPs, and the
size of GMPs is about 25 nm in diameter. The GMPs and sodium citrate were also measured through the FT-IR spectra, as shown in Fig. 2D. The 1530 cm−1 peak attributed to the C O stretching vibration from the COOH group of citrate shifts to an intense band at about 1525 cm−1 for GMPs. It demonstrated citrate’s bonding on the surface of GMPs by chemisorptions. 3.2. Characterizations of the nanoparticle-Apt2 conjugates Nanoparticles, aptamer and NPs-Apts conjugates were characterized by UV–vis spectroscopy and the results are shown in Fig. 3. The formation of conjugates was characterized by the UV–vis absorption spectrum. Fig. 3a, c and e recorded the UV–vis absorption of GNPs, MNPs and GMPs respectively. After coated with Apt2, UV–vis curves showed red shifts for all three particles. The bare GNPs exhibited a characteristic absorption peak at 518 nm (Fig. 3a), which related to the surface plasmon resonance of nanosized Au. After coated with Apt2, UV–vis absorption was observed at 520 nm due to the formation of GNPs-Apt2 (curve b). Curve c shows the UV–vis absorption spectrum of MNPs with an absorption peak at 223 nm. After the incubation of Apt2, the absorption peak shifts to 247 nm, indicating that Apt2 has been successful associated to MNPs. Curves e and f show the absorption peak of GMPs at 548 nm and new absorption peak of GMPs-Apt2 at 559 nm, it also demonstrate the Apt2 was successful bound onto GMPs. The curve g is the UV–vis absorption spectrum of Apt2 and the absorption peak of Apt2 was appeared at 260 nm, which correspond to the UV–vis absorption peak of DNA. All these results verify that Apt2 has been immobilized on the nanoparticles successfully.
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Fig. 3. UV–vis spectra of GNPs (a, black), GNPs-Apt2 conjugates (b, red), MNPs (c, blue), MNPs-Apt2 conjugates (d, green), GMPs (e, pink), GMPs-Apt2 conjugates (f, dark green) and Apt2 (g, dark blue). (For interpretation of the color information in this figure legend, the reader is referred to the web version of the article.)
3.3. Immobilization of Apt1 on gold chip surface Apt1 was immobilized on the gold surface by self-assembly via the covalent bond between Au and the thiol group of aptamer as described elsewhere [26]. SPR was employed to characterize the
Fig. 4. Sensorgram of SPR angle shifts according to the immobilization of Apt1 and MCH on chip surface.
immobilization. The interaction between Au and Apt1 was monitored by observing changes in the resonant angle. The SPR angle increase in millidegrees is used as a response unit to quantify the binding of macromolecules to the sensor surface. A change of 120 millidegrees represents a surface change of approximately 1 ng mm−2 as described in previous reports [27–29]. To obtain well aligned aptamer monolayers [30], 1 mM MCH was injected into two
Fig. 5. The sensogram of SPR signal amplification processes with three kinds of nanoparticles GNPs (A), MNPs (B), GMPs (C) and histogram of the comparison of three nanoparticles (D). The black line is experimental group. The red line is control group, which is treated with BSA instead of thrombin.
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Fig. 6. AFM images of the bare gold film (A) and sequentially treated with Apt1/thrombin/GNPs-Apt2 (B), Apt1/thrombin/MNPs-Apt2 (C), and Apt1/thrombin/GMPs-Apt2 (D).
channels for 1 h. As shown in Fig. 4, an increase in the SPR angle was observed, the adsorption of aptamer on SPR gold chip results in a big angle shift of 789 millidegrees. The surface coverage of aptamer on the gold chip is 6.575 ng mm−2 as calculated. 3.4. SPR signal enhancement by three NPs-Apt2 Aptamer prefers to adopt G-quarter structure when binding with thrombin [31]. Incubation conditions, such as pH value, temperature and so on, have an impact on the formation of the G-quarter structure [32]. Thrombin was dissolved in Tris buffer (pH 6.2, 100 mM MES, 50 mM Tris, 40 mM KCl, 0.05% Triton X-100, and 1% DMSO) and injected into the SPR chamber for 30 min at room temperature (Fig. 5, experimental group, black line). In order to confirm the specific binding of Apt1 and thrombin, a two channel SPR system was used. The control group (Fig. 5, red line) was injected with bovine serum albumin (BSA) solution with the corresponding concentrations. As shown in Fig. 5, the SPR angle was increased evidently with the injection of thrombin. However, when BSA with same concentration was injected into the SPR chamber, no obvious SPR signal was observed. This result confirms that the Apt1 has a sufficient specificity and thrombin can be identified
with high selectivity. After the incubation of thrombin, the conjugate of nanoparticles and Apt2 was injected into SPR chamber for 15 min. The increase of SPR angle is obvious difference with the injection of different NPs-Apt2 conjugates. Fig. 5 shows SPR angel shifts after the injection of GNPs-Apt2, MNPs-Apt2 and GMPsApt2 conjugates are 313, 431and 604 millidegrees, respectively. The amplification GMPs-Apt2 conjugate for SPR signal is 1.93 and 1.40 times of that of GNPs-Apt2 and MNPs-Apt2 respectively. However, even after the injection of NPs-Apt2 into the control group, the SPR signal is kept stable. These results demonstrate the formation of Apt1/thrombin/NPs-Apt2 sandwich structure successfully. As shown in Fig. 5D, compared with GNPs and MNPs, the signal amplification of GMPs for SPR spectroscopy is more sensitive. This enhancement was mainly attributed by the larger mass and higher refractive index of the GMP nanoparticles. Therefore, we choose GMPs-Apt2 conjugates as signal amplification element in following thrombin detection. To further confirm the binding of NPs-Apt2 onto the thrombin modified chip surface, we carried out AFM image characterization. Fig. 6A shows the surface geometry of the bare gold film. After the immobilization of Apt1 and the formation of sandwich structure, the images reveal that the height increased from 5 to 35 nm
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Fig. 7. Direct detection of thrombin (A) and its comparison with amplification strategy using GMPs at variable concentrations of thrombin (B).
(Fig. 6B–D). Among three NPs, GNP and MNP show high aggregation which may due to the lower stability after modified with Apt2. The monodispersed GMP shows good uniform and lower aggregation on the sensor chip surface.
great potential of GMPs in the field of protein detection and disease diagnosis.
Acknowledgements 3.5. Thrombin detection via Apt-immobilized GMPs The SPR detection of thrombin was preformed to evaluate the sensing performance of the Apt-immobilized GMPs as amplification reagent. It could be clearly seen from the curves in Fig. 7A that the SPR angel shifts gradually increase with the concentration of thrombin concentration increasing from 10 to 100 nM. However, when the concentration of thrombin concentration is lower than 10 nM, it is very difficult to discriminate the concentration of thrombin by SPR spectroscopy due to its low molecular weight. Obviously, this detection limit is not enough for the detection of thrombin due to the trace amount of thrombin existed in serum. In order to detect thrombin at low concentrations, GMPs-Apt2 conjugates were used as an amplification reagent. As shown in Fig. 7B, SPR angel shifts resulted in the binding of GMPs-Apt2 conjugates greatly increase with the thrombin concentrations in the range of 0.1–100 nM (black curve). Thrombin detection using GMPs-Apt2 conjugates for signal enhancement shows good linearity from 0.1 to 100 nM with R2 = 0.98. The limit of detection (LOD) was calculated to be 0.60 nM (LOD = 3.3 × standard deviation/slope). At the concentration of 100 nM, the SPR angle shift is enlarged for 5 times comparing with that of control group without GMPs-Apt2. Therefore, the increase in the SPR signal obtained for the experimental group can definitely own to GMPs-Apt2 conjugates. The whole trends of the experimental group and the control group verified that GMPs is an outstanding means of signal amplification for SPR. Aptamer modified GMPs can result in significant enhancement of the SPR sensor’s sensitivity.
4. Conclusions In summary, the monodispersed core/shell Fe3 O4 /Au magnetic nanoparticles (GMPs) have been synthesized. To evaluate the practicability of using GMPs in enhancing SPR signal for biosensing, thrombin is used as the model analyte to construct SPR-based sandwich biosensor. The experimental results demonstrate that the addition of GMPs-Apt2 conjugates results in a big SPR angle change, indicating that GMPs possess of the ability of enhancing the sensitivity of SPR sensor as an amplification reagent. It shows
This work is supported by the National Natural Science Foundation of China (Grant Nos. 61275085, 31100560).
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Biographies Hongxia Chen received her B.S. degree in chemistry in 1996 from Zhengzhou University, China, and MS degree in 2003 and Ph.D. in 2008 from Pusan National University, Korea. Dr. Chen is an associate professor in School of Life Sciences, Shanghai University. Her current research interests include optical sensors, biochip, bioanalytical nanochemistry and nanotoxicology. Fangjie Qi received his B.S. degree from school of Kewen in 2012 from Jiangsu Normal University, China. He is a master course student in School of Life Sciences, Shanghai University. His current research interests are biosensor and cancer cell detection. Hongjian Zhou received his B.S. degree in chemistry in 2007 from Nanyang Institute of Technology, China, and MS degree in Materials physics and chemistry in 2010 from Jiangsu University of Science and Technology, China. He got his Ph.D. degree in Nano Fusion Technology in 2014 from Pusan National University. Now he is a Korean National Research Foundation (KNRF)-funded research fellow in Department of Cogno-Mechatronics Engineering, Pusan National University. His current research interest includes synthesis of multifunctional nanoparticles and biosensor applications. He is also interested in surface modification of nanocomposites for biocompatibility and biodegradability in bio-nano-medical applications. Shengsong Jia received her B.S. degree in 2013 from School of Xinke, Hennan Institude of Science and Technology, China. She is a master course student in School of Life Sciences, Shanghai University. Her current research interests are biosensor and small molecule detection. Yanmin Gao received her B.S. degree in 2013 from school of biological science, Nanyang Normal University, China. She is a master course student in School of Life Sciences, Shanghai University. Her current research interests are biosensor and cancer cell detection. Kwangnak Koh received M.S. degree in 1992 from Pusan National University and Ph.D. in 1995 in supramolecular engineering from Kyushu University, Japan. He is a professor in institute of general education, Pusan National University, Korea. His research interests include biochip, supramolecular engineering, bioanalytical nanochemistry and bionanomaterials. Yongmei Yin received her bachelor, master and Ph.D. degree at Nanjing Medical University in 1991, 2001 and 2008, respectively. She is currently the vice-Dean of the First Affiliated Hospital of Nanjing Medical University, and the vice-director of the Department of Oncology at this Hospital.